ANALYSIS OF THE APHAKIA GENE REQUIRED FOR LENS FORMATION

描述先天性或后天性的眼透镜缺陷如白内障是视力受损和失明的主要原因。本研究提案的长期目标是通过理解透镜发育和维持的分子机制来改进透镜疾病的诊断、治疗甚至预防。拟议的研究集中在分离和分析的基因，这是特别需要的透镜发展的小鼠。常染色体隐性遗传无晶状体（ak）突变纯合子小鼠由于缺少透镜、虹膜和瞳孔而失明，伴有小眼症。在胚胎发育过程中，最早的缺陷部分随机化的纺锤体方向在有丝分裂的上皮细胞的透镜基板，以及在相关的细胞外基质（EM）的不规则性组合的位置克隆和位置候选基因的方法将被用来确定基因突变的ak。含有ak基因座的染色体间隔将通过使用来自种间杂交的纯合后代的多态性标记的连锁分析来精细作图。将使用现有的YAC重叠群作为起点构建基因座的高分辨率物理图谱，通过构建BAC克隆重叠群进一步细化，然后进行外显子捕获或cDNA选择以鉴定候选基因。在适当的发育阶段分离的RNA将用于评估这些基因在正常和猴眼中的表达，并生成用于通过SSCP和DNA序列分析进行突变筛选的cDNA。此外，为了获得关于EM的组成和作用的更精确的信息，EM紧密地连接视杯与透镜基板，表达，已知EM成分的表达将通过免疫定位和原位杂交技术在正常和突变眼中进行研究。

DESCRIPTION Congenital or acquired defects of the ocular lens such as cataract are a major cause of impaired vision and blindness. The long-term objective of this research proposal is an improved diagnosis, treatment or even prevention of lens disease by means of although understanding of the molecular mechanisms underlying development and maintenance of the lens. The proposed study is focused on isolation and analysis of a gene that is specifically required for lens development in the mouse. Mice homozygous for the autosomal recessive aphakia (ak) mutation are blind due to a absence of lens, iris and pupil with microphtalmia. During embryonic development, the earliest defects a partial randomization of spindle orientation in mitotic epithelial cells of the lens placode, as well as irregularities in the associated extracellular matrix (EM) A combined positional cloning and positional candidate gene approach will be used to identify the gene mutated in ak. The chromosomal interval containing the ak locus will be fine mapped by linkage analysis with polymorphic markers using homozygous offspring derived from an inter-specific cross. A high- resolution physical map of the locus will be constructed using existing YAC contigs as a starting point, further refined by construction of a BAC clone contig, and followed by exon trapping or cDNA selection to identify candidate genes. RNA isolated at appropriate developmental stages will be used to assess the expression of these genes in the normal and monkey eyes, and to generate cDNA to be used in mutation screening by SSCP and DNA sequence analysis. Furthermore,, to gain more precise information about the composition and role of the EM that intimately connects the optic cup with the lens placode, the expression, the expression of known EM constituents will be studied by immunolocalization and in situ hybridization techniques in normal and mutant eyes.